DIY Gentra Puregene Protocol

Description

There was a company called Gentra, they made a nice extraction kit (salt style). Qiagen bought them out, and then their kit became very expensive. Not to worry, some dudes put the old kits through a GCMS and reverse engineered it.

What you need

1. Ammonium acetate
   
   

Protocol

1. Take tissue, smash it in the ball mill (or preferred method)


2. Add 500µl of Gentra Lysis Buffer (below) and 4µl proK and lyse @ 55^oC for 3 hrs or overnight


3. Add 2µl RNAse (10mg/ml) to each sample


4. Incubate at 37^oC for 30mins


5. Cool to RT, add 500µl Protein Precipitation Buffer (7.5M Ammonium Acetate)


6. Vortex vigorously, then ice for 15min


7. Spin full speed for 3min, remove supernatant unless protein pellet not tight (spin again). Transfer spernatant to new tube.


8. Add equal volume (500µl) 100% EtoH or isopropanol


9. Spin max speed 15mins, tip out ethanol (not pellet).


10. Add 500µl 70% EtOH


11. Spin 5 mins full speed


12. Dry ethanol off


13. Resuspend in favourite buffer (reccomend T-BUF, 10mM tris ph8)

Lysis Buffer 1l (10mM Tris, 100mMEDTA, 2% SDS, ph8)

Protein Precipitation buffer (7.5 Ammonium Acetate)